Photocleavable fluorescent nucleotides for DNA sequencing on chip constructed by site-specific coupling chemistry

ABSTRACT

This invention provides a method for determining the sequence of a DNA or an RNA, wherein (i) about 1000 or fewer copies of the DNA or RNA are bound to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii) each copy of the DNA or RNA comprises a self-priming moiety.

This application is a §371 national stage of PCT InternationalApplication No. PCT/US2005/006960, filed Mar. 3, 2005, and claims thebenefit of U.S. provisional application no. 60/550,007, filed Mar. 3,2004, the contents of all of which are hereby incorporated by referenceinto this application.

The invention disclosed herein was made with Government support underCenter of Excellence in Genomic Science grant No. P50 HG002806 from theNational Institutes of Health, U.S. Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

Throughout this application, various publications are referenced inparentheses by number. Full citations for these references may be foundat the end of each experimental section. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application to more fully describe the state of the art towhich this invention pertains.

BACKGROUND

DNA sequencing is a fundamental tool for biological science. Thecompletion of the Human Genome Project has set the stage for screeninggenetic mutations to identify disease genes on a genome-wide scale (1).Accurate high-throughput DNA sequencing methods are needed to explorethe complete human genome sequence for applications in clinical medicineand health care. Recent studies have indicated that an important routefor identifying functional elements in the human genome involvessequencing the genomes of many species representing a wide sampling ofthe evolutionary tree (2). To overcome the limitations of the currentelectrophoresis-based sequencing technology (3-5), a variety of newDNA-sequencing methods have been investigated. Such approaches includesequencing by hybridization (6), mass spectrometry based sequencing(7-9), and sequence-specific detection of single-stranded DNA usingengineered nanopores (10). More recently, DNA sequencing by synthesis(SBS) approaches such as pyrosequencing (11), sequencing of single DNAmolecules (12) and polymerase colonies (13) have been widely explored.

The concept of DNA sequencing by synthesis was revealed in 1988 (14).This approach involves detection of the identity of each nucleotideimmediately after its incorporation into a growing strand of DNA in apolymerase reaction. Thus far, no complete success has been reported inusing such a system to sequence DNA unambiguously. An SBS approach usingphotocleavable fluorescent nucleotide analogues on a surface wasproposed in 2000 (15). In this approach, modified nucleotides are usedas reversible terminators, in which a different fluorophore with adistinct fluorescent emission is linked to each of the 4 bases through aphotocleavable linker and the 3′-OH group is capped by a small chemicalmoiety. DNA polymerase incorporates only a single nucleotide analoguecomplementary to the base on a DNA template covalently linked to asurface. After incorporation, the unique fluorescence emission isdetected to identify the incorporated nucleotide and the fluorophore issubsequently removed photochemically. The 3′-OH group is then chemicallyregenerated, which allows the next cycle of the polymerase reaction toproceed. Since the large surface on a DNA chip can have a high densityof different DNA templates spotted, each cycle can identify many basesin parallel, allowing the simultaneous sequencing of a large number ofDNA molecules. The advantage of using photons as reagents for initiatingphotoreactions to cleave the fluorophore is that no additional chemicalreagents are required to be introduced into the system and cleanproducts can be generated with no need for subsequent purification.

SUMMARY

This invention provides a method for determining the sequence of a DNA,wherein (i) about 1000 or fewer copies of the DNA are bound to a solidsubstrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii)each copy of the DNA comprises a self-priming moiety, comprisingperforming the following steps for each nucleic acid residue of the DNAto be sequenced:

-   -   (a) contacting the bound DNA with DNA polymerase and four        photocleavable fluorescent nucleotide analogues under conditions        permitting the DNA polymerase to catalyze DNA synthesis,        wherein (i) the nucleotide analogues consist of an analogue of        G, an analogue of C, an analogue of T and an analogue of A, so        that a nucleotide analogue complementary to the residue being        sequenced is bound to the DNA by the DNA polymerase, and (ii)        each of the four analogues has a pre-determined fluorescence        wavelength which is different than the fluorescence wavelengths        of the other three analogues;    -   (b) removing unbound nucleotide analogues; and    -   (c) determining the identity of the bound nucleotide analogue,    -   thereby determining the sequence of the DNA.

This invention also provides a method for determining the sequence of anRNA, wherein (i) about 1000 or fewer copies of the RNA are bound to asolid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and(ii) each copy of the RNA comprises a self-priming moiety, comprisingperforming the following steps for each nucleic acid residue of the RNAto be sequenced:

-   -   (a) contacting the bound RNA with RNA polymerase and four        photocleavable fluorescent nucleotide analogues under conditions        permitting the RNA polymerase to catalyze RNA synthesis,        wherein (i) the nucleotide analogues consist of an analogue of        G, an analogue of C, an analogue of U and an analogue of A, so        that a nucleotide analogue complementary to the residue being        sequenced is bound to the RNA by the RNA polymerase, and (ii)        each of the four analogues has a pre-determined fluorescence        wavelength which is different than the fluorescence wavelengths        of the other three analogues;    -   (b) removing unbound nucleotide analogues; and    -   (c) determining the identity of the bound nucleotide analogue,    -   thereby determining the sequence of the RNA.

This invention also provides a composition of matter comprising a solidsubstrate having a DNA bound thereto via 1,3-dipolar azide-alkynecycloaddition chemistry, wherein (i) about 1000 or fewer copies of theDNA are bound to the solid substrate, and (ii) each copy of the DNAcomprises a self-priming moiety.

This invention also provides a composition of matter comprising a solidsubstrate having an RNA bound thereto via 1,3-dipolar azide-alkynecycloaddition chemistry, wherein (i) about 1000 or fewer copies of theRNA are bound to the solid substrate, and (ii) each copy of the RNAcomprises a self-priming moiety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: DNA extension reaction performed in solution phase tocharacterize the 4 different photocleavable fluorescent nucleotideanalogues (dUTP-PC-R6G, dGTP-PC-Bodipy-FL-510, dATP-PC-ROX,dCTP-PC-Bodipy-650). After each extension reaction, the DNA extensionproduct (SEQ ID NOs. 1-4) is purified by HPLC for MALDI-TOF MSmeasurement to verify that it is the correct extension product.Photolysis is performed to produce a DNA product that is used as aprimer for the next DNA extension reaction.

FIG. 2. Polymerase extension scheme. Primer extended with dUTP-PC-R6G(1), and its photocleavage product 2; Product 2 extended withdGTP-PC-Bodipy-FL-510 (3), and its photocleavage product 4; Product 4extended with dATP-PC-ROX (5), and its photocleavage product 6; Product6 extended with dCTP-PC-Bodipy-650 (7), and its photocleavage product 8.After 10 seconds of irradiation with a laser at 355 nm, photocleavage iscomplete with all the fluorophores cleaved from the extended DNAproducts.

FIG. 3. Panels (1)-(8). MALDI-TOF MS spectra of the four consecutiveextension products and their photocleavage products. Primer extendedwith dUTP-PC-R6G (1), and its photocleavage product 2; Product 2extended with dGTP-PC-Bodipy-FL-510 (3), and its photocleavage product4; Product 4 extended with dATP-PC-ROX (5), and its photocleavageproduct 6; Product 6 extended with dCTP-PC-Bodipy-650 (7), and itsphotocleavage product 8. After 10 seconds of irradiation with a laser at355 nm, photocleavage is complete with all the fluorophores cleaved fromthe extended DNA products.

FIG. 4. Immobilization of an azido-labeled PCR product on analkynyl-functionalized surface and a ligation reaction between theimmobilized single-stranded DNA template and a loop primer to form aself-priming DNA moiety on the chip. The sequence of the loop primer isshown in (A).

FIG. 5. Schematic representation of SBS on a chip using four PCfluorescent nucleotides (Upper panel) and the scanned fluorescenceimages for each step of SBS on a chip (Lower panel). (1) Incorporationof dATP-PC-ROX; (2) Photocleavage of PC-ROX; (3) Incorporation ofdGTP-PC-Bodipy-FL-510; (4) Photocleavage of PC-Bodipy-FL-510; (5)Incorporation of dATP-PC-ROX; (6) Photocleavage of PC-ROX; (7)Incorporation of dCTP-PC-Bodipy-650; (8) Photocleavage of PC-Bodipy-650;(9) Incorporation of dUTP-PC-R6G; (10) Photocleavage of PC-R6G; (11)Incorporation of dATP-PC-ROX; (12) Photocleavage of PC-ROX; (13)Incorporation of dUTP-PC-R6G; (14) Photocleavage of PC-R6G; (15)Incorporation of dATP-PC-ROX; (16) Photocleavage of PC-ROX; (17)Incorporation of dGTP-PC-Bodipy-FL-510; (18) Photocleavage ofPC-Bodipy-FL-510; (19) Incorporation of dUTP-PC-R6G; (20) Photocleavageof PC-R6G; (21) Incorporation of dCTP-PC-Bodipy-650; (22) Photocleavageof PC-Bodipy-650; (23) Incorporation of dATP-PC-ROX; (24) Photocleavageof PC-ROX.

FIG. 6. Structures of dGTP-PC-Bodipy-FL-510 (λ_(abs (max))=502 nm;λ_(em (max))=510 nm), dUTP-PC-R6G (λ_(abs (max))=525 nm;λ_(em (max))=550 nm), dATP-PC-ROX (λ_(abs (max))=575 nm;λ_(em (max))=602 nm), and dCTP-PC-Bodipy-650 (λ_(abs (max))=630 nm;λ_(em (max))=650 nm)

FIG. 7. Synthesis of photocleavable fluorescent nucleotides. (a)acetonitrile or DMF/1 M NaHCO₃ solution; (b) N,N′-disuccinimidylcarbonate (DSC), triethylamine; (c) 0.1 M Na₂CO₃/NaHCO₃ aqueous buffer(pH 8.5-8.7).

DETAILED DESCRIPTION OF THE INVENTION

Terms

The following definitions are presented as an aid in understanding thisinvention:

A Adenine; C Cytosine; DNA Deoxyribonucleic acid; G Guanine; RNARibonucleic acid; SBS Sequencing by synthesis; T Thymine; and U Uracil.

“Nucleic acid” shall mean any nucleic acid, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996 1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

As used herein, “self-priming moiety” shall mean a nucleic acid moietycovalently bound to a nucleic acid to be transcribed, wherein the boundnucleic acid moiety, through its proximity with the transcriptioninitiation site of the nucleic acid to be transcribed, permitstranscription of the nucleic acid under nucleic acidpolymerization-permitting conditions (e.g. the presence of a suitablepolymerase, nucleotides and other reagents). That is, the self-primingmoiety permits the same result (i.e. transcription) as does a non-boundprimer. In one embodiment, the self-priming moiety is a single strandednucleic acid having a hairpin structure. Examples of such self-primingmoieties are shown in the Figures.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid based on sequence complementarity. Thepropensity for hybridization between nucleic acids depends on thetemperature and ionic strength of their milieu, the length of thenucleic acids and the degree of complementarity. The effect of theseparameters on hybridization is well known in the art (see Sambrook J,Fritsch E F, Maniatis T. 1989. Molecular cloning: a laboratory manual.Cold Spring Harbor Laboratory Press, New York.)

As used herein, “nucleotide analogue” shall mean an analogue of A, G, C,T or U which is recognized by DNA or RNA polymerase (whichever isapplicable) and incorporated into a strand of DNA or RNA (whichever isappropriate). Examples of nucleotide analogues include, withoutlimitation 7-deaza-adenine, 7-deaza-guanine, the analogues ofdeoxynucleotides shown in FIG. 6, analogues in which a label is attachedthrough a cleavable linker to the 5-position of cytosine or thymine orto the 7-position of deaza-adenine or deaza-guanine, analogues in whicha small chemical moiety such as —CH₂OCH₃ or —CH₂CH═CH₂ is used to capthe —OH group at the 3′-position of deoxyribose, and analogues ofrelated dideoxynucleotides. Nucleotide analogues, includingdideoxynucleotide analogues, and DNA polymerase-based DNA sequencing arealso described in U.S. Pat. No. 6,664,079.

Embodiments of the Invention

This invention provides a method for determining the sequence of a DNA,wherein (i) about 1000 or fewer copies of the DNA are bound to a solidsubstrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii)each copy of the DNA comprises a self-priming moiety, comprisingperforming the following steps for each nucleic acid residue of the DNAto be sequenced:

-   -   (a) contacting the bound DNA with DNA polymerase and four        photocleavable fluorescent nucleotide analogues under conditions        permitting the DNA polymerase to catalyze DNA synthesis,        wherein (i) the nucleotide analogues consist of an analogue of        G, an analogue of C, an analogue of T and an analogue of A, so        that a nucleotide analogue complementary to the residue being        sequenced is bound to the DNA by the DNA polymerase, and (ii)        each of the four analogues has a pre-determined fluorescence        wavelength which is different than the fluorescence wavelengths        of the other three analogues;    -   (b) removing unbound nucleotide analogues; and    -   (c) determining the identity of the bound nucleotide analogue,    -   thereby determining the sequence of the DNA.

In one embodiment, the instant method further comprises the step ofphotocleaving the fluorescent moiety from the bound nucleotide analoguefollowing step (c).

In another embodiment of the instant method, the solid substrate isglass or quartz.

In a further embodiment of the instant method, fewer than 100 copies ofthe DNA, fewer than 20 copies of the DNA, or fewer than five copies ofthe DNA are bound to the solid substrate.

In still a further embodiment, one copy of the DNA is bound to the solidsubstrate.

This invention also provides a method for determining the sequence of anRNA, wherein (i) about 1000 or fewer copies of the RNA are bound to asolid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and(ii) each copy of the RNA comprises a self-priming moiety, comprisingperforming the following steps for each nucleic acid residue of the RNAto be sequenced:

-   -   (a) contacting the bound RNA with RNA polymerase and four        photocleavable fluorescent nucleotide analogues under conditions        permitting the RNA polymerase to catalyze RNA synthesis,        wherein (i) the nucleotide analogues consist of an analogue of        G, an analogue of C, an analogue of U and an analogue of A, so        that a nucleotide analogue complementary to the residue being        sequenced is bound to the RNA by the RNA polymerase, and (ii)        each of the four analogues has a pre-determined fluorescence        wavelength which is different than the fluorescence wavelengths        of the other three analogues;    -   (b) removing unbound nucleotide analogues; and    -   (c) determining the identity of the bound nucleotide analogue,    -   thereby determining the sequence of the RNA.

In one embodiment the instant method, further comprises the step ofphotocleaving the fluorescent moiety from the bound nucleotide analoguefollowing step (c).

In another embodiment of the instant method, the solid substrate isglass or quartz.

In a further embodiment of the instant method, fewer than 100 copies ofthe RNA, fewer than 20 copies of the RNA, or fewer than five copies ofthe RNA are bound to the solid substrate.

In still a further embodiment, one copy of the RNA is bound to the solidsubstrate.

This invention also provides a composition of matter comprising a solidsubstrate having a DNA bound thereto via 1,3-dipolar azide-alkynecycloaddition chemistry, wherein (i) about 1000 or fewer copies of theDNA are bound to the solid substrate, and (ii) each copy of the DNAcomprises a self-priming moiety.

This invention also provides a composition of matter comprising a solidsubstrate having an RNA bound thereto via 1,3-dipolar azide-alkynecycloaddition chemistry, wherein (i) about 1000 or fewer copies of theRNA are bound to the solid substrate, and (ii) each copy of the RNAcomprises a self-priming moiety.

In one embodiment of the instant methods and compositions of matter, thenumber of DNA or RNA copies bound to the solid substrate exceeds 1000,and can be, for example, about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or greater.

This invention also provides a compound having the structure:

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

Synopsis

Here, the procedure for performing SBS on a chip using a synthetic DNAtemplate and photocleavable pyrimidine nucleotides (C and U) isdisclosed (also see 16). In addition, the design and synthesis of 4photocleavable nucleotide analogues (A, C, G, U), each of which containsa unique fluorophore with a distinct fluorescence emission is described.Initially, it was established that these nucleotides are good substratesfor DNA polymerase in a solution-phase DNA extension reaction and thatthe fluorophore can be removed with high speed and efficiency by laserirradiation (λ˜355 nm). SBS was then performed using these 4photocleavable nucleotide analogues to identify the sequence of a DNAtemplate immobilized on a chip. The DNA template was produced by PCRusing an azido-labeled primer, and was immobilized on the surface of thechip with 1,3-dipolar azide-alkyne cycloaddition chemistry. Aself-priming moiety was then covalently attached to the DNA template byenzymatic ligation to allow the polymerase reaction to proceed on theDNA immobilized on the surface. This is the first report of using acomplete set of photocleavable fluorescent nucleotides for 4-color DNAsequencing by synthesis.

Introduction

A 4-color DNA sequencing by synthesis (SBS) on a chip using fourphotocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510,dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) is disclosed herein.Each nucleotide analogue consists of a different fluorophore attached tothe 5-position of the pyrimidines (C and U) and the 7-position of thepurines (G and A) through a photocleavable 2-nitrobenzyl linker. Afterverifying that these nucleotides could be successfully incorporated intoa growing DNA strand in a solution-phase polymerase reaction and thefluorophore could be cleaved using laser irradiation (λ˜355 nm) in 10seconds, an SBS reaction was performed on a chip which contained aself-priming DNA template covalently immobilized using 1,3-dipolarazide-alkyne cycloaddition. The DNA template was produced by apolymerase chain reaction using an azido-labeled primer and theself-priming moiety was attached to the immobilized DNA template byenzymatic ligation. Each cycle of SBS consisted of the incorporation ofthe photocleavable fluorescent nucleotide into the DNA, detection of thefluorescent signal and photocleavage of the fluorophore. The entireprocess was repeated to identify 12 continuous bases in the DNAtemplate. These results demonstrate that photocleavable fluorescentnucleotide analogues can be incorporated accurately into a growing DNAstrand during a polymerase reaction in solution phase as well as on achip. Moreover, all 4 fluorophores can be detected and then efficientlycleaved using near-UV irradiation, thereby allowing continuousidentification of the DNA template sequence.

To demonstrate the feasibility of carrying out DNA sequencing bysynthesis on a chip, four photocleavable fluorescent nucleotideanalogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, anddCTP-PC-Bodipy-650) (FIG. 6) were synthesized according to the schemeshown in FIG. 7 using a similar procedure as reported previously (16).Modified DNA polymerases have been shown to be highly tolerant tonucleotide modifications with bulky groups at the 5-position ofpyrimidines (C and U) and the 7-position of purines (A and G) (17, 18).Thus, each unique fluorophore was attached to the 5 position of C/U andthe 7 position of A/G through a photocleavable 2-nitrobenzyl linker.

In order to verify that these fluorescent nucleotides are incorporatedaccurately in a base-specific manner in a polymerase reaction, fourcontinuous steps of DNA extension and photocleavage by near UVirradiation were carried out in solution as shown in FIG. 1. This allowsthe isolation of the DNA product at each step for detailed molecularstructure characterization as shown in FIG. 2. The first extensionproduct 5′-U(PC-R6G)-3′ 1 was purified by HPLC and analyzed usingMALDI-TOF MS [FIG. 3]. This product was then irradiated at 355 nm usingan Nd-YAG laser for 10 seconds and the photocleavage product 2 was alsoanalyzed using MALDI-TOF MS [FIG. 3]. Near UV light absorption by thearomatic 2-nitrobenzyl linker causes reduction of the 2-nitro group to anitroso group and an oxygen insertion into the carbon-hydrogen bondfollowed by cleavage and decarboxylation (19). As can be seen from FIG.3, Panel 1, the MALDI-TOF MS spectrum consists of a distinct peak at m/z6536 corresponding to the DNA extension product 5′-U(PC-R6G)-3′ (1),which confirms that the nucleotide analogue can be incorporated basespecifically by DNA polymerase into a growing DNA strand. The small peakat m/z 5872 corresponding to the photocleavage product is due to thepartial cleavage caused by the nitrogen laser pulse (337 nm) used inMALDI ionization. For photocleavage, a Nd-YAG laser was used toirradiate the DNA product carrying the fluorescent nucleotide for 10seconds at 355 nm to cleave the fluorophore from the DNA extensionproduct. FIG. 3, Panel 2, shows the photocleavage result of the aboveDNA product. The peak at m/z 6536 has completely disappeared while thepeak corresponding to the photocleavage product 5′-U (2) appears as thesole dominant peak at m/z 5872, which establishes that laser irradiationcompletely cleaves the fluorophore with high speed and efficiencywithout damaging the DNA. The next extension reaction was carried outusing this photocleaved DNA product as a primer along withdGTP-PC-Bodipy-FL-510 to yield an extension product5′-UG(PC-Bodipy-FL-510)-3′ (3). As described above, the extensionproduct 3 was purified, analyzed by MALDI-TOF MS producing a dominantpeak at m/z 6751 [FIG. 3, Panel 3], and then photocleaved for further MSanalysis yielding a single peak at m/z 6255 (product 4) [FIG. 3, Panel4]. The third extension using dATP-PC-ROX to yield 5′-UGA(PC-ROX)-3′(5), the fourth extension using dCTP-PC-Bodipy-650 to yield5′-UGAC(PC-Bodipy-650)-3′ (7) and their photocleavage to yield products6 and 8 were similarly carried out and analyzed by MALDI-TOF MS as shownin FIG. 3, Panels 5-8. These results demonstrate that theabove-synthesized four photocleavable fluorescent nucleotide analoguescan successfully incorporate into the growing DNA strand in a polymerasereaction, and the fluorophore can be efficiently cleaved by near UVirradiation, which makes it feasible to use them for SBS on a chip.

The photocleavable fluorescent nucleotide analogues were then used in anSBS reaction to identify the sequence of the DNA template immobilized ona solid surface as shown in FIG. 4. A site-specific 1,3-dipolarcycloaddition coupling chemistry was used to covalently immobilize theazido-labeled double-stranded PCR products on the alkynyl-functionalizedsurface in the presence of a Cu(I) catalyst. Previously, we have shownthat DNA is successfully immobilized on the glass surface by thischemistry and evaluated the functionality of the surface-bound DNA andthe stability of the array using a primer extension reaction (16). Thesurface-immobilized double stranded PCR product was denatured using a0.1 M NaOH solution to remove the complementary strand without the azidogroup, thereby generating a single-stranded PCR template on the surface.Then, a 5′-phosphorylated self-priming moiety (loop primer) was ligatedto the 3′-end of the above single stranded DNA template using Taq DNAligase (20). The structure of the loop primer was designed to bear athermally stable loop (21) and stem sequence with a melting temperatureof 89° C. The 12-bp overhanging portion of the loop primer was madecomplementary to the 12-bp sequence of the template at its 3′ end toallow the Taq DNA ligase to seal the nick between the 5′-phosphate groupof the loop primer and the 3′-hydroxyl group of the single-stranded DNAtemplate. This produces a unique DNA moiety that can self-prime for thesynthesis of a complementary strand. The ligation was found to be inquantitative yield in a parallel solution-phase reaction using the sameprimer and single-stranded DNA template.

The principal advantage offered by the use of a self-priming moiety ascompared to using separate primers and templates is that the covalentlinkage of the primer to the template in the self-priming moietyprevents any possible dissociation of the primer from the template undervigorous washing conditions. Furthermore, the possibility of mis-primingis considerably reduced and a universal loop primer can be used for allthe templates allowing enhanced accuracy and ease of operation. SBS wasperformed on the chip-immobilized DNA template using the 4photocleavable fluorescent nucleotide analogues, see FIG. 5. Thestructure of the self-priming DNA moiety is shown schematically in theupper panel, with the first 12 nucleotide sequence immediately after thepriming site. The sequencing reaction on the chip was initiated byextending the self-priming DNA using dATP-PC-ROX (complementary to the Ton the template), and Thermo Sequenase DNA polymerase. After washing,the extension of the primer by a single fluorescent nucleotide wasconfirmed by observing an orange signal (the emission signal from ROX)in a microarray scanner [FIG. 5, (1)]. After detection of thefluorescent signal, the surface was irradiated at 355 nm for 1 min usingan Nd-YAG laser to cleave the fluorophore. The surface was then washed,and a negligible residual fluorescent signal was detected to confirmcomplete photocleavage of the fluorophore [FIG. 5, (2)]. This wasfollowed by incorporation of the next fluorescent nucleotidecomplementary to the subsequent base on the template. The entire processof incorporation, detection and photocleavage was performed multipletimes using the four photocleavable fluorescent nucleotide analogues toidentify 12 successive bases in the DNA template. The integratedfluorescence intensity on the spot, obtained from the scanner software,indicated that the incorporation efficiency was over 90% and more than97% of the original fluorescence signal was removed by photocleavage. Anegative control experiment consisting of incubating the self-primingDNA moiety with dATP-PC-ROX in the absence of DNA polymerase and washingthe surface showed that negligible fluorescence remained as compared tothat of FIG. 5, (1).

In summary, four photocleavable fluorescent nucleotide analogues havebeen synthesized and characterized and have been used to produce 4-colorDNA sequencing data on a chip. These nucleotides have been shown to beexcellent substrates for the DNA polymerase and the fluorophore could becleaved efficiently using near UV irradiation. This is important withrespect to enhancing the speed of each cycle in SBS for high throughputDNA analysis. Also, a PCR-amplified DNA template can be ligated with aself-priming moiety demonstrated that and its sequence can be accuratelyidentified in a DNA polymerase reaction on a chip, indicating that a PCRproduct from any organism can be potentially used as a template for theSBS system in the future. The modification of the 3′-OH of thephotocleavable fluorescent nucleotide with a small chemical group toallow reversible termination may be considered. The library ofphotocleavable fluorescent nucleotides reported here will alsofacilitate single molecule DNA sequencing approaches.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich unless otherwiseindicated. ¹H NMR spectra were recorded on a Bruker 400 spectrometer.High-resolution MS (HRMS) data were obtained by using a JEOL JMS HX 110Amass spectrometer. Mass measurement of DNA was made on a Voyager DEmatrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)mass spectrometer (Applied Biosystems). Photolysis was performed using aSpectra Physics GCR-150-30 Nd-YAG laser that generates light pulses at355 nm (ca. 50 mJ/pulse, pulse length ca. 7 ns) at a frequency of 30 Hzwith a light intensity at ca. 1.5 W/cm². The scanned fluorescenceemission images were obtained by using a ScanArray Express scanner(Perkin-Elmer Life Sciences) equipped with four lasers with excitationwavelengths of 488, 543, 594, and 633 nm and emission filters centeredat 522, 570, 614, and 670 nm.

Synthesis of Photocleavable Fluorescent Nucleotides

Photocleavable fluorescent nucleotides dGTP-PC-Bodipy-FL-510,dUTP-PC-R6G, dATP-PC-ROX and dCTP-PC-Bodipy-650 (FIG. 6) weresynthesized according to FIG. 7 using a similar method as reported (16).A photocleavable linker (PC-Linker)1-[5-(aminomethyl)-2-nitrophenyl]ethanol was reacted with the NHS esterof the corresponding fluorescent dye to produce an intermediate PC-Dye,which was converted to a PC-Dye NHS ester by reacting with N,N′-disuccinimidyl carbonate. The coupling reaction between the differentPC-Dye NHS esters and the amino nucleotides (dATP-NH₂ and dGTP-NH₂ fromPerkin-Elmer; dUTP-NH₂ from Sigma; dCTP-NH₂ from TriLinkBioTechnologies) produced the 4 photocleavable fluorescent nucleotides.

The synthesis of the photocleavable fluorescent nucleotidedCTP-PC-Bodipy-650 was reported (Seo, T. S., Bai, X., Ruparel, H., Li,Z., Turro, N. J. & Ju, J. (2004) Proc. Natl. Acad. Sci. USA 101, 54885493). dUTP-PC-R6G, dGTP-PC-Bodipy-FL-510 and dATP-PC-ROX were preparedwith a similar procedure as shown in FIG. 7.

A general procedure for the synthesis of PC—Dye:1-[5-(Aminomethyl)-2-nitrophenyl]ethanol (2) (PC-Linker, 5 mg, 26 μmol)was dissolved in 550 μl of acetonitrile (for Bodipy) or DMF (for R6G andROX) and then mixed with 100 μl of 1 M NaHCO3 aqueous solution. Asolution of the NHS ester of the corresponding fluorophore (MolecularProbes) (13 μmol) in 400 pl of acetonitrile or DMF was added slowly tothe above reaction mixture and then stirred for 5 h at room temperature.The resulting reaction mixture was purified on a preparative silica-gelTLC plate (CHCl₃/CH₃OH, 4/1 for Bodipy, 1/1 for R6G and ROX) to yieldpure PC-Dye. PC-R6G: (92% yield) ₁NMR (400 z, CD₃ D) 8.17 (d, 2H),7.87(d, 2H),7.76 (d, 1H), 7.45(d, 1H), 7.04 (s, 2H), 6.89 (s, 2H), 5.34(q, 1H), 4.70 (s, 2H), 3.52(q, 4H), 2.14 (s, 6H), 1.47(d, 3H), 1.35 (t,6H). HRMS (FAB₊) m/z: calcd for C₃₆H₃₇N₄O₇ (M+H₊), 637.2622; found,637.2643. PC-ROX: (90% yield) ₁NMR (400 z, CD₃ D) 8.11 (d, 2H), 7.88 (m,2H), 7.69 (d, 1H), 7.45 (dd, 1H), 6.79(s, 1H), 5.36(q, 1H), 4.69 (s,2H), 3.50 (m, 8H), 3.08(t, 4H), 2.73 (t, 4H), 2.11 (t, 4H), 1.95 (t,4H), 1.47 (d, 3H). HRMS (FAB₊) m/z: calcd for C₄₂H₄₁N₄O₇ (M+H₊),713.2975; found, 713.2985. PC-Bodipy-FL-510 was reported (Li, Z., Bai,X., Ruparel, H., Kim, S., Turro, N. J. & Ju, J. (2003) Proc. Natl. Acad.Sci. USA 100, 414-419.)

A general procedure for the synthesis of PC-Dye NHS ester:N,N′-disuccinimidyl carbonate (4.27 mg, 17 μmol) and triethylamine (4.6μl, 33 μmol) were added to a solution of PC-Dye (11 μmol) in 200 μl ofdry acetonitrile or DMF. The reaction mixture was stirred under argon atroom temperature for 6 h. The solvent was removed under vacuum, and theresidue was immediately purified by flash column chromatography(CH₂Cl₂/CH₃OH, 4/1). PC-R6G NHS ester: (28% yield) ₁H NMR (400 MHz,CD₃OD) 8.15 (s, 2H), 8.03 (d, 1H), 7.78 (m, 1H), 7.71 (d, 1H), 7.56 (dd,2H), 7.02 (m, 2H), 6.86 (m, 2H), 6.30 (q, 1H), 4.78 (d, 1H), 4.67 (d,1H), 3.51 (q, 4H), 2.67 (s, 4H), 2.12 (s, 6H), 1.73 (d, 3H), 1.35 (t,6H). HRMS (FAB₊) m/z: calculated for C₄₁H₄₀O₁₁N₅ (M+H₊), 778.2724;found, 778.2731. PC-ROX NHS ester: (35% yield) ₁H NMR3 (400 MHz, CD₃OD)8.09 (m, 2H), 8.02 (d, 1H), 7.69-7.75(m, 2H), 7.54 (dd, 2H), 6.77(m,2H), 6.30 (q, 1H), 4.78 (d, 1H), 4.66 (d, 1H), 3.47-3.57 (m, 8H),3.04-3.10 (m, 4H), 2.64-2.72 (m, 8H), 2.06-2.14 (m, 4H), 1.90-1.98 (m,4H), 1.74 (d, 3H). HRMS (FAB₊) m/z: calcd for C₄₇H₄₄O₁₁N₅ (M+H₊),854.3037; found, 854.3069. PC-Bodipy-FL-510 NHS ester was reported (Li,Z., Bai, X., Ruparel, H., Kim, S., Turro, N. J. & Ju, J. (2003) Proc.Natl. Acad. Sci. USA 100, 414-419.)

A general procedure for the synthesis of photocleavable fluorescentnucleotides dUTP-PCR6G, dGTP-PC-Bodipy-FL-510 and dATP-PC-ROX

PC-Dye NHS ester (30 μmol) in 300 μl of acetonitrile or DMF was added toa solution of amino nucleotide dNTP-NH₂ (1 μmol) in 300 μl of 0.1 MNa₂CO₃—NaHCO₃ buffer (pH 8.5-8.7). The reaction mixture was stirred atroom temperature for 3 h. A preparative silica-gel TLC plate was used toseparate the unreacted PCDye NHS ester from the fractions containingfinal photocleavable fluorescent nucleotides (CHCl₃/CH₃OH, 1/1). Theproduct was concentrated further under vacuum and purified withreverse-phase HPLC on a 150 4.6-mm C18 column to obtain the pureproduct. Mobile phase: A, 8.6 mM triethylamine/100 mMhexafluoroisopropyl alcohol in water (pH 8.1); B, methanol. Elution wasperformed with 100% A isocratic over 10 min followed by a lineargradient of 0-50% B for 20 min and then 50% B isocratic over another 20min.

DNA Polymerase Reaction using 4 Photocleavable Fluorescent NucleotideAnalogues in Solution

The four nucleotide analogues, dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G,dATP-PC-ROX and dCTP-PC-Bodipy-650 were characterized by performing fourcontinuous DNA-extension reactions sequentially using a primer(5′-AGAGGATCCAACCGAGAC-3′) (SEQ ID NO:5) and a synthetic DNAtemplate(5′-GTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGAT-CCTCTATTGTGTCCGG-3′)(SEQ ID NO:6) corresponding to a portion of exon 7 of the human p53 gene(FIG. 1). The four nucleotides in the template immediately adjacent tothe annealing site of the primer were 3′-ACTG-5′. First, a polymeraseextension reaction using dUTP-PC-R6G along with the primer and thetemplate was performed producing a single base extension product. Thereaction mixture for this, and all subsequent extension reactions,consisted of 80 pmol of template, 50 pmol of primer, 80 pmol of theparticular photocleavable fluorescent nucleotide, 1× Thermo Sequenasereaction buffer, and 4 U of Thermo Sequenase DNA polymerase (AmershamBiosciences) in a total volume of 20 μL. The reaction consisted of 25cycles at 94° C. for 20 sec, 48° C. for 40 sec, and 60° C. for 75 sec.Subsequently, the extension product was purified by using reverse-phaseHPLC. An Xterra MS C18 (4.6×50-mm) column (Waters) was used for the HPLCpurification. Elution was performed over 120 minutes at a flow rate of0.5 mL/min with the temperature set at 50° C. by using a linear gradient(12-34.5%) of methanol in a buffer consisting of 8.6 mM triethylamineand 100 mM hexafluoroisopropyl alcohol (pH 8.1). The fraction containingthe desired DNA product was collected and freeze-dried for analysisusing MALDI-TOF mass spectrometry. For photocleavage, the purified DNAextension product bearing the fluorescent nucleotide analogue wasresuspended in 200 μL of deionized water. The mixture was irradiated for10 seconds in a quartz cell with path lengths of 1.0 cm employing aNd-YAG laser at 355 nm and then analyzed by MALDI-TOF MS. Afterphotocleavage, the DNA product with the fluorophore removed was used asa primer for a second extension reaction using dGTP-PC-Bodipy-FL-510.The second extended product was then purified by HPLC and photolyzed.The third extension using dATP-PC-ROX and the fourth extension usingdCTP-PC-Bodipy-650 were carried out in a similar manner using thepreviously extended and photocleaved product as the primer.

PCR Amplification to Produce Azido-Labeled DNA Template

An azido-labeled PCR product was obtained using a 100-bp template(5′-AGCGACTGCTATCATGTCATATCGACGTGCTCACTAGCTCTACATATGCGTGCGTGATCAGATGACGTATCGATGCTGACTATAGTCTCCCATGCGAGTG-3′), (SEQ IDNO:7) a 24-bp azido-labeled forward primer(5′-N₃-AGCGACTGCTATCATGTCATATCG-3′), (SEQ ID NO:8) and a 24-bp unlabeledreverse primer (5′-CACTCGCATGGGAGACTATAGTCA-3′). (SEQ ID NO: 9) In atotal reaction volume of 50 μL, 1 pmol of template and 30 pmol offorward and reverse primers were mixed with 1 U of AccuPrime Pfx DNApolymerase and 5 μL of 10× AccuPrime Pfx reaction mix (Invitrogen)containing 1 mM of MgSO₄ and 0.3 mM of dNTP. The PCR reaction consistedof an initial denaturation step at 95° C. for 1 min, followed by 38cycles at 94° C. for 15 sec, 63° C. for 30 sec, 68° C. for 30 sec. Theproduct was purified using a 96 QlAquick multiwell PCR purification kit(Qiagen) and the quality was checked using 2% agarose gelelectrophoresis in 1× TAE buffer. The concentration of the purified PCRproduct was measured using a Perkin-Elmer Lambda 40 UV-Visspectrophotometer.

Construction of a Self-Priming DNA Template on a Chip by EnzymaticLigation

The amino-modified glass slide (Sigma) was functionalized to contain aterminal alkynyl group as described previously (16). The azido-labeledDNA product generated by PCR was dissolved in DMSO/H₂O (1/3, v/v) toobtain a 20 μM solution. 5 μL of the DNA solution was mixed with CuI (10nmol, 100 eq.) and N,N-diisopropyl-ethylamine (DIPEA) (10 nmol, 100 eq.)and then spotted onto the alkynyl-modified glass surface in the form of6 μL drops. The glass slide was incubated in a humid chamber at roomtemperature for 24 hr, washed with deionized water (dH₂O) and SPSCbuffer (50 mM sodium phosphate, 1 M NaCl, pH 6.5) for 1 hr (16), andfinally rinsed with dH₂O. To denature the double stranded PCR-amplifiedDNA to remove the non-azido-labeled strand, the glass slide was immersedinto 0.1 M NaOH solution for 10 min and then washed with 0.1 M NaOH anddH₂O, producing a single stranded DNA template that is immobilized onthe chip. For the enzymatic ligation of a self-priming moiety to theimmobilized DNA template on the chip, a 5′-phosphorylated 40-bp loopprimer (5′-PO3-GCTGAATTCCGCGTTCGCGGAATTCAGCCACTCGCATGGG-3′) (SEQ IDNO:10) was synthesized. This primer contained a thermally stable loopsequence 3′-G(CTTG)C-5′, a 12-bp stem, and a 12-bp overhanging end thatwould be annealed to the immobilized single stranded template at its3′-end. A 10 μL solution consisting of 100 pmol of the primer, 10 U ofTaq DNA ligase, 0.1 mM NAD, and 1× reaction buffer (New England Biolabs)was spotted onto a location of the chip containing the immobilized DNAand incubated at 45° C. for 4 hr. The glass slide was washed with dH₂O,SPSC buffer, and again with dH₂O. The formation of a stable hairpin wasascertained by covering the entire surface with 1× reaction buffer (26mM TrisHCl/6.5 mM MgCl₂, pH 9.3), incubating it in a humid chamber at94° C. for 5 min to dissociate any partial hairpin structure, and thenslowly cooling down to room temperature for reannealing.

SBS reaction on a Chip with Four Photocleavable Fluorescent NucleotideAnalogues

One microliter of a solution consisting of dATP-PC-ROX (60 pmol), 2 U ofThermo Sequenase DNA polymerase, and 1× reaction buffer was spotted onthe surface of the chip, where the self-primed DNA moiety wasimmobilized. The nucleotide analogue was allowed to incorporate into theprimer at 72° C. for 5 min. After washing with a mixture of SPSC buffer,0.1% SDS, and 0.1% Tween 20 for 10 min, the surface was rinsed with dH₂Oand ethanol successively, and then scanned with a ScanArray Expressscanner to detect the fluorescence signal. To perform photocleavage, theglass chip was placed inside a chamber (50×50×50 mm) filled withacetonitrile/water (1/1, v/v) solution and irradiated for 1 min with theNd-YAG laser at 355 nm. The light intensity applied on the glass surfacewas ca. 1.5 W/cm². After washing the surface with dH₂O and ethanol, thesurface was scanned again to compare the intensity of fluorescence afterphotocleavage with the original fluorescence intensity. This process wasfollowed by the incorporation of dGTP-PC-Bodipy-FL-510, with thesubsequent washing, fluorescence detection, and photocleavage processesperformed as described above. The same cycle was repeated 10 more timesusing each of the four photocleavable fluorescent nucleotide analoguescomplementary to the base on the template. For a negative controlexperiment, 1 μL solution containing dATP-PC-ROX (60 pmol), and 1×reaction buffer was added on to the DNA immobilized on the chip in theabsence of DNA polymerase and then incubated at 72° C. for 5 min,followed by the same washing and detection steps as above.

REFERENCES

-   -   1. Collins, F. S., Green, E. D., Guttmacher, A. E. &        Guyer, M. S. (2003) Nature 422, 835-847.    -   2. Thomas, J. W., Touchman, J. W., Blakesley, R. W.,        Bouffard, G. G., Beckstrom-Sternberg, S. M., Margulies, E. H.,        Blanchette, M., Siepel, A. C., Thomas, P. J. & McDowell, J. C.        et al. (2003) Nature 424, 788-793.    -   3. Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P.,        Dodd, C., Connell, C. R., Heiner, C., Kent, S. B. H. &        Hood, L. E. (1987) Nature 321, 674-679.    -   4. Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N. &        Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351.    -   5. Doherty, E. A. S., Kan, C. W. and Barron, A. E. (2003)        Electrophoresis, 24, 4170-4180.    -   6. Drmanac, S., Kita, D., Labat, I., Hauser, B., Schmidt, C.,        Burczak, J. D. & Drmanac, R. (1998) Nat. Biotechnol. 16, 54-58.    -   7. Fu, D. J., Tang, K., Braun, A., Reuter, D., Darnhofer-Demar,        B., Little, D. P., O'Donnell, M. J., Cantor, C. R. &        Koster, H. (1998) Nat. Biotechnol. 16, 381-384.    -   8. Roskey, M. T., Juhasz, P., Smirnov, I. P., Takach, E. J.,        Martin, S. A. & Haff, L. A. (1996) Proc. Natl. Acad. Sci. USA        93, 4724-4729.    -   9. Edwards, J. R., Itagaki, Y. & Ju, J. (2001) Nucleic Acids        Res. 29, e104 (p1-6).    -   10. Kasianowicz, J. J., Brandin, E., Branton, D. &        Deamer, D. W. (1996) Proc. Natl. Acad. Sci. USA 93, 13770-13773.    -   11. Ronaghi, M., Uhlen, M. & Nyren, P. (1998) Science 281,        363-365.    -   12. Braslavsky, I., Hebert, B., Kartalov, E. &        Quake, S. R. (2003) Proc. Natl. Acad. Sci. USA 100, 3960-3964.    -   13. Mitra, R. D., Shendure, J., Olejnik, J., Olejnik, E. K. &        Church, G. M. (2003) Anal. Biochem. 320, 55-65.    -   14. Hyman, E. D. (1988) Anal. Biochem. 174, 423-436.    -   15. Ju, J., Li, Z., Edwards, J. & Itagaki, Y. (2003) U. S. Pat.        No. 6,664,079.    -   16. Seo, T. S., Bai, X., Ruparel, H., Li, Z., Turro, N. J. &        Ju, J. (2004) Proc. Natl. Acad. Sci. USA, 101, 5488-5493.    -   17. Rosenblum, B. B., Lee, L. G., Spurgeon, S. L., Khan, S. H.,        Menchen, S. M., Heiner, C. R. & Chen, S. M. (1997) Nucleic Acids        Res. 25, 4500-4504.    -   18. Zhu, Z., Chao, J., Yu, H. & Waggoner, A. S. (1994) Nucleic        Acids Res. 22, 3418-3422    -   19. Rajasekharan Pillai, V. N. (1980) Synthesis 1, 1-26. 20.        Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189-193.    -   21. Antao, V. P., Lai, S. Y. & Tinoco, I. Jr. (1991) Nucleic        Acids Res. 19, 5901-5905.

1. A method for determining the sequence of a DNA, wherein (i) 1000 orfewer copies of the DNA are bound to a solid substrate via 1,3-dipolarazide-alkyne cycloaddition chemistry and (ii) each copy of the DNA is adenatured single-stranded template and comprises a 5′-phosphorylatedself-priming moiety covalently linked to a 3′-end of the DNA, comprisingperforming the following steps: (a) contacting the bound DNA with a DNApolymerase and four photocleavable fluorescent nucleotide analoguesunder conditions permitting the DNA polymerase to catalyze DNAsynthesis, wherein (i) the nucleotide analogues consist of an analogueof G, an analogue of C, an analogue of T and an analogue of A, so that anucleotide analogue complementary to the residue of the single-strandedtemplate being sequenced is incorporated into the DNA extension producton the 5′-phosphorylated self-priming moiety of the bound DNA by the DNApolymerase, and (ii) each of the four analogues has a pre-determinedfluorescence emission wavelength which is different than thefluorescence emission wavelengths of the other three analogues; (b)removing the unincorporated nucleotide analogues not incorporated intothe DNA extension product on the 5′-phosphorylated self-priming moietyof the bound DNA; (c) determining the identity of the incorporatednucleotide analogue and; (d) repeating steps (a) to (c) forincorporation of the next nucleotide analogue complementary to thesubsequent base of the bound single stranded template DNA, therebydetermining the sequence of the DNA.
 2. The method of claim 1, furthercomprising the step of photocleaving the fluorescent moiety from theincorporated nucleotide analogue following step (c).
 3. The method ofclaim 1, wherein the solid substrate is glass or quartz.
 4. The methodof claim 1, wherein fewer than 100 copies of the DNA are bound to thesolid substrate.
 5. The method of claim 1, wherein fewer than 20 copiesof the DNA are bound to the solid substrate.
 6. The method of claim 1,wherein fewer than five copies of the DNA are bound to the solidsubstrate.
 7. The method of claim 1, wherein one copy of the DNA isbound to the solid substrate.
 8. A method for determining the sequenceof an RNA, wherein (i) 1000 or fewer copies of the RNA are bound to asolid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and(ii) each copy of the RNA is a single-stranded template and comprises a5′-phosphorylated self-priming moiety covalently linked to a 3′-end ofthe bound RNA, comprising performing the following steps: (a) contactingthe bound RNA with a RNA polymerase and four photocleavable fluorescentnucleotide analogues under conditions permitting the RNA polymerase tocatalyze RNA synthesis, wherein (i) the nucleotide analogues consist ofan analogue of G, an analogue of C, an analogue of U and an analogue ofA, so that a nucleotide analogue complementary to the residue of thesingle-stranded template being sequenced is incorporated into the RNAextension product on the 5′-phosphorylated self-priming moiety of thebound RNA by the RNA polymerase, and (ii) each of the four analogues hasa pre-determined fluorescence emission wavelength which is differentthan the fluorescence emission wavelengths of the other three analogues;(b) removing the unincorporated nucleotide analogues not incorporatedinto the RNA extension product on the 5′ phosphorylated self-primingmoiety of the bound RNA; (c) determining the identity of theincorporated nucleotide analogue and; (d) repeating steps (a) to (c) forincorporation of the next nucleotide analogue complementary to thesubsequent base of the bound single stranded template RNA, therebydetermining the sequence of the RNA.
 9. The method of claim 8, furthercomprising the step of photocleaving the fluorescent moiety from theincorporated nucleotide analogue following step (c).
 10. The method ofclaim 8, wherein the solid substrate is glass or quartz.
 11. The methodof claim 8, wherein fewer than 100 copies of the RNA are bound to thesolid substrate.
 12. The method of claim 8, wherein fewer than 20 copiesof the RNA are bound to the solid substrate.
 13. The method of claim 8,wherein fewer than five copies of the RNA are bound to the solidsubstrate.
 14. The method of claim 8, wherein one copy of the RNA isbound to the solid substrate.
 15. A composition of matter comprising asolid substrate having a DNA or an RNA bound thereto via 1,3-dipolarazide-alkyne cycloaddition chemistry, wherein (i) about 1000 or fewercopies of the DNA or the RNA are bound to the solid substrate, and (ii)each copy of the DNA or the RNA comprises a self-priming moiety.
 16. Acompound having the structure:


17. The compound of claim 16 having the having the structure:


18. A composition of matter comprising a solid substrate having a RNAbound thereto via 1,3-dipolar azide-alkyne cycloaddition chemistry,wherein (i) about 1000 or fewer copies of the RNA are bound to the solidsubstrate, and (ii) each copy of the RNA comprises a self-primingmoiety.
 19. The compound of claim 16 having the structure:


20. The compound of claim 16 having the structure:


21. The compound of claim 16 having the structure: